Trifolium pratense L. (red clover) extract and doxorubicin synergistically inhibits proliferation of 4T1 breast cancer in tumor-bearing BALB/c mice through modulation of apoptosis and increase antioxidant and anti-inflammatory related pathways

Therapeutic strategies against triple-negative breast cancer (TNBC) are associated with drug-induced toxicities. The tropical edible red clover (Trifolium pratense L.) is rich in polyphenolic compounds which confer the plant potential anticancer properties. The aim of this study was to investigate the effects of T. pretense and doxorubicin (DOX) on the apoptosis and proliferation of 4T1 tumor cells in an allograft model of tumor-bearing BALB/c mice. Fifty-six female 4T1-tumor bearing- BALB/c mice were randomly divided into 7 groups (n = 8/group) to receive different doses and combinations of DOX and T. pretense extract for 35 days. On the 36th day, serum estradiol (E2), IL-12 and IFN-gamma cytokines, and glutathione peroxidase (GPx) activity were measured. Tumor's ferric reducing antioxidant power (FRAP) and the expressions of apoptosis-related genes (p53, Bax, Bcl-2, and caspase-3) were also evaluated. Immunohistochemical staining for Ki-67 and p53 were performed. Our results showed that the co-treatment of DOX and T. pretense (100-400 mg/kg) inhibited the proliferation of 4T1 tumor cells in dose- and time-dependent manners. The cotreatment of DOX and T. pretense (especially at the dose of 400 mg/kg) decreased the serum level of E2 (as a stimulant for breast tumor growth) and increased the serum levels of IL-12 and IFN-gamma along with significant increments in serum GPx and tumor FRAP activities. The co-administration of DOX and T. pretense also decreased the expression of Ki-67 proliferation marker and increased the number p53 positive (i.e., apoptotic) cells within tumors. This was accompanied with the upregulation of proapoptotic and down-regulation of antiapoptotic genes. The key findings indicated the synergistic effects of DOX and T. pretense against TNBC xenografts.